Summary The overall goal of the U19 proposal is to combine kidney and islet transplantation with nonmyeloablative hematopoietic cell transplantation (HCT) to achieve immune tolerance to the donor tissues. We will use a the cynomolgus monkey model to develop this approach for deceased and living donor scenarios, using expanded polyclonal and donor-specific recipient regulatory T cells respectively, in Projects 1 and 2. We hypothesize that tolerance will involve both expansion and induction of donor-specific regulatory T cells and deletion of donor- specific effector T cells. A key to development of this procedure will be the tools to identify and track these donor-specific T cells. We previously developed such a method in humans. Core B will extend this technology to cynomolgus monkeys (cyno) and apply the assay to support Projects 1 and 2. Specifically, Core B will follow the same development pipeline we have used for human and mouse to develop a T cell receptor (TCR) sequencing assay for cyno. Since TCRs rearrange somatically with massive diversity, each TCR is nearly unique. This allows us to track hundreds of thousands of T cell clones over time and between tissues by their TCR sequence. We leverage with technology by first isolating donor-specific T cells through a mixed lymphocyte reaction (explicitly stimulating T cells that are specific to the donor tissue). The clones in these expanded donor- reactive CD4 and CD8 T cells are compared to those in sorted, unstimulated functional subsets, including effector and regulatory T cells, and the expanded sequences are identified as belonging to the donor-reactive subset. We will validate this assay in cynomolgus monkeys receiving allotransplants and optimize methods .for defining donor-reactive effector and regulatory T cell clones. Timed, sorted T cell DNA samples from infused Tregs, peripheral blood and graft-infiltrating lymphocytes as well as DNA from biopsy specimens and urine pellets from transplanted cyno monkeys in Projects 1 and 2 will be sent to Core B, where the assay will be applied. Over time, we will monitor the clonal expansion and contraction of these T cell subsets in blood, tissue, and urine pellets to identify mechanisms of immune tolerance in this preclinical model.